Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa

An increase in scrotal temperature can lead to the production of poor quality spermatozoa and infertility. In the present study we have used mice to examine the impact of mild, scrotal heat stress (42 degrees C for 30 min) on numbers of spermatozoa as well as on the integrity of their DNA. Spermatozoa recovered from the epididymides hours (1 to 24) or days (7 to 32) after treatment were analysed using COMET and sperm chromatin structure (SCSA) assays. The treatment induced a stress response in both the testis and the epididymis that was associated with reduced expression of the cold inducible RNA binding protein (Cirp) and an increase in germ cell apoptosis (Apotag positive cells). Although spermatozoa present in the epididymis at the time of heating contained correctly packaged DNA, its integrity was compromised by heat stress. In addition, although some germ cells, which were present within the testis at the time of heat stress, were removed by apoptosis, many germ cells completed their development and were recovered as motile spermatozoa with damaged DNA. In conclusion, these data demonstrate that scrotal heat stress can compromise the DNA integrity of spermatozoa and this may have clinical implications for patients undergoing IVF and intra-cytoplasmic sperm injection (ICSI).     

Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse

Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen-thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milk + 0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14 + PI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P < 0.05-0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P < 0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P < 0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm.     

Effects of scrotal insulation on viability characteristics of cryopreserved bovine semen.

The effect of a 48-h scrotal insulation on spermatozoal viability (motility and acrosomal integrity), before and after semen cryopreservation, was studied in six young Holstein bulls whose semen was collected twice in succession at 3-d intervals. Motility and acrosomal integrity were measured before and after incubation of semen at 37 degrees C for 3 h. For assessment of results, collection days were grouped: period 1 (control) = d -6, -3, and 0, where d 0 = initiation of scrotal insulation after semen collection; period 2 = d 3, 6, and 9 (sperm presumed in the epididymis or rete testis during scrotal insulation); period 3 = d 12, 15, ... 39 (sperm presumed in spermatogenesis during scrotal insulation). Semen was cryopreserved each collection day until morphologically abnormal cells exceeded 50% of the ejaculate (d 12 to 21). Semen viability before and after freezing was lower in period 3 than in period 1 (P less than .05). These differences coincided with the appearance in period 3 of abnormal sperm morphology and depressed undiluted semen motility, which began on d 12 (P less than .01). Semen collected during period 2 that was extended but unfrozen did not differ from that collected during period 1 in morphology or viability. However, for frozen semen, period 2 was significantly poorer than period 1 for both viability measurements, but only after incubation for 3 h at 37 degrees C postthaw (P less than .05). We conclude that epididymal sperm are adversely affected by elevated testicular temperatures, as noted by their decreased ability to maintain motility and acrosomal integrity following cryopreservation.     

Improvement of semen quality by nocturnal scrotal cooling and moderate behavioural change to reduce genital heat stress in men with oligoasthenoteratozoospermia

A questionnaire assessing factors that might cause an increase in scrotal temperature was completed by patients with reproducible oligoasthenoteratozoospermia of idiopathic nature or caused by varicocele. Evaluation by means of a grading scale revealed increased scrotal heat stress in oligoasthenoteratozoospermic patients compared with normozoospermic men (P < 0.01). In addition, long-term determination of 24 h scrotal temperature profiles showed that compared with semen donors, oligoasthenoteratozoospermic patients frequently had scrotal temperatures above 35.5 degrees C despite the same environmental temperatures (P < 0.05). In 88% of cases, maximum scrotal temperatures were measured during rest or sleep phases, whereas minimum values were recorded during physical activity or frequent change of position. Nocturnal scrotal cooling by means of an air stream resulted in a decrease in scrotal temperature of approximately 1 degrees C. Furthermore, a highly significant increase in sperm concentration (P < 0.0001) and total sperm output (P < 0.0001) was achieved after nocturnal scrotal cooling for 12 weeks together with a moderate decrease in factors leading to genital heat stress. A significant improvement in sperm motility (P < 0.05) and sperm morphology (P < 0.05) was also observed, but this improvement was markedly less pronounced than the changes in sperm concentration. This study shows the importance of genital heat stress as a cofactor in fertility impairment in men and indicates nocturnal scrotal cooling as a therapeutic option.     

Developmental competence and oxidative state of mouse zygotes heat-stressed maternally or in vitro

Mammalian preimplantation embryos are sensitive to maternal and direct heat stress. However, the mechanisms by which heat stress affects early embryonic development in vivo or in vitro are unknown. This study examined whether heat-stress-induced loss of developmental competence in mouse embryos was mediated by physiological changes in the maternal environment or by high temperatures alone. After fertilization, zygotes at the same stage were heat-stressed at 39.5 degrees C for 12 h either maternally (measured by maternal rectal temperature) or directly in culture. Zygotes in each group were cultured at 37.5 degrees C for a further 84 h to assess their developmental ability. Neither type of heat stress affected the first cleavage rate. However, the proportion of embryos that developed to morulae or blastocysts was significantly lower in the maternally heat-stressed group, but not in the directly heat-stressed group. Moreover, maternal heat stress significantly reduced intracellular glutathione concentrations and enhanced hydrogen peroxide concentrations in both zygotes and two-cell embryos that were recovered immediately after heat stress or 12 h later, respectively. In contrast, direct heat stress had little effect on concentrations of glutathione or hydrogen peroxide in cultured early embryos. These results demonstrate that maternal heat stress at the zygote stage reduces the developmental ability of mouse embryos via physiological changes in the maternal environment that lead to an increase in intracellular oxidative stress on the embryo.     

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice

Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin beta(C), increased. Inhibin/activin beta(A) and beta(B) subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the beta(A) subunit also detected in peritubular myoid cells. The alpha, beta(A), beta(B) and beta(C) subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While beta(A) and beta(B) subunit proteins were observed in spermatogonia and spermatocytes in immature testes, beta(C) was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear beta(A) subunit protein was observed in primary spermatocytes and nuclear beta(C) subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.     

Reduced embryonic survival in rainbow trout resulting from paternal exposure to the environmental estrogen 17alpha-ethynylestradiol during late sexual maturation

Exposure of fishes to environmental estrogens is known to affect sexual development and spawning, but little information exists regarding effects on gametes. This study evaluated embryonic survival of offspring from male rainbow trout (Oncorhynchus mykiss) exposed to 17alpha-ethynylestradiol (EE(2)) using an in vitro fertilization protocol. Males were exposed at either 1800 or 6700 degree days ( degrees d) (i.e. 161 or 587 days post-fertilization (dpf)) to test for effects on testes linked to reproductive ontogeny. At 1800 degrees d, fish were beginning testicular differentiation and were exposed to 109 ng EE(2)/l for 21 days. At 6700 degrees d, fish have testes containing spermatocytes and spermatids and were exposed for 56 days to either 0.8, 8.3, or 65 ng EE(2)/l. Semen was collected at full sexual maturity in each group and used to fertilize eggs pooled from several non-exposed females. Significant decreases in embryonic survival were observed only with the 6700 degrees d exposure. In 0.8 and 8.3 ng EE(2)/l treatments, embryo survival was significantly reduced at 19 dpf when compared with the control. In contrast, an immediate decrease in embryonic survival at 0.5 dpf was observed in the 65 ng EE(2)/l treatment. Blood samples collected at spawning from 6700 degrees d exposed males revealed a significant decrease in 11-ketotestosterone and a significant increase in luteinizing hormone levels for the 65 ng EE(2)/l treatment when compared with the other treatment groups. Results indicate that sexually maturing male rainbow trout are susceptible to EE(2) exposure with these fish exhibiting two possible mechanisms of reduced embryonic survival through sperm varying dependant on EE(2) exposure concentrations experienced.     

Postnatal testicular development in mouse species with different levels of sperm competition

Postcopulatory sexual selection leads to an increase in sperm numbers which is partly the result of an increase in relative testes mass and could also be the consequence of changes in testis architecture or function. Very little is known regarding developmental changes during the first spermatogenic wave that may lead to enhanced spermatogenic efficiency and increased sperm production. We examined testicular development after birth in four mouse species with different sperm competition levels to assess changes in testicular architecture and function. Differences in relative testes mass between species appeared soon after birth and were exacerbated thereafter. The volume of testes occupied by seminiferous tubules differed between species postnatally and were associated with sperm competition levels. Finally, changes over time in the proportions of tubules with different germ cell types were also associated with sperm competition levels, with the time taken for the transition between various cell stages being negatively associated with levels of sperm competition. We conclude that postnatal testis development differs between closely related species with different sperm competition levels influencing testis architecture and the rate of progression of spermatogenesis, leading to differences in testis function at reproductive maturity.     

Spermatogenesis and steroidogenesis in mouse,hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts

Retrieval, extracorporal storage and autotransplantation of testicular tissue could become an important strategy for preserving male gonadal function. The present study used syngeneic and immunodeficient nude mice as hosts, and immature and adult mice, neonatal and adult photoregressed Djungarian hamsters and neonatal marmosets to identify the potential of testicular tissue grafting to maintain the morphological and functional integrity of the testis. Testicular tissue was grafted s.c. either as fresh tissue or after cryopreservation into adult, orchidectomized hosts. The mice that received rodent testis tissue were autopsied 50 days later, and blood samples were collected. Sixty-five per cent of mouse isografts contained morphologically normal testicular tissue and seminiferous tubules with some degree of spermatogenic recovery. Mature spermatozoa were recovered after enzymatic disaggregation. Although the recovery of spermatogenesis was limited in adult mouse and hamster tissue, complete spermatogenesis was observed in grafts from immature rodents. Testicular tissue from neonatal marmosets developed up to the stage of spermatocytes at day 135 after xenografting. Androgen concentrations were comparable in intact control mice and in mice receiving fresh mouse and hamster grafts, slightly lower in mice receiving cryopreserved grafts and adult photoregressed hamster tissue, and low in castrated control mice and in mice receiving marmoset tissue. These results show that isografts and xenografts of immature and adult testicular tissue become functionally active as a s.c. graft in the mouse and that this approach might be useful in combination with cryopreservation as a tool for storage and activation of the male germ line and androgen replacement therapy in patients.     

DL-alpha-tocopherol acetate mitigates maternal hyperthermia-induced pre-implantation embryonic death accompanied by a reduction of physiological oxidative stress in mice

Maternal hyperthermia induces pre-implantation embryo death, which is accompanied by enhanced physiological oxidative stress. We evaluated whether the administration of DL-alpha-tocopherol acetate (TA) to hyperthermic mothers mitigated pre-implantation embryo death. Mice were exposed to heat stress (35 degrees C, 60% relative humidity) for 12 h or not heated (25 degrees C) on the day of mating. Twelve hours before the beginning of temperature treatment, TA was injected intraperitoneally at a dose of 1 g/kg body weight. After the treatment, zygotes were recovered and the developmental abilities and intracellular glutathione (GSH) levels were evaluated. Another set of mice, with or without TA treatment, was exposed to heat stress for 12, 24 and 36 h, and the urinary levels of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured. Heat stress significantly decreased the blastocyst development rate and the GSH content in zygotes, as compared with the non-heat-stressed embryos, while TA administration significantly mitigated the deleterious effects of heat stress with regard to both parameters. Moreover, although the urinary levels of 8-OHdG gradually increased according to the duration of heat exposure, with or without TA administration, the levels were lower in the TA-administered group than in the placebo-injected mice. These results suggest that heat stress enhances physiological oxidative stress, and that TA administration alleviates the hyperthermia-induced death of pre-implantation embryos by reducing physiological oxidative stress.     

Sperm in poor quality semen from bulls during heat stress have a lower affinity for binding hydrogen-3 heparin

Binding assays with [3H] heparin were performed using spermatozoa collected prior to, during, and following summer heat stress to dairy bulls. Ejaculates collected in August 1983 after a period of ambient temperatures exceeding 29.4 degrees C exhibited a high frequency of abnormal sperm, and motility was reduced in some samples. Sperm in samples collected during heat stress possessed dissociation constants for binding [3H] heparin ranging from 134.5 to 163.2 nmol. In contrast, sperm in semen collected prior to and after heat stress had significantly lower dissociation constants (higher affinity) for [3H] heparin, 12.9 to 56.4 nmol. The number of binding sites for [3H] heparin on sperm did not change among collection periods. It was concluded that the binding affinity for [3H] heparin may reflect membrane integrity of bull sperm.     

Short-term storage of cane toad (Bufo marinus) gametes

The responses of cane toad (Bufo marinus) gametes, used as a model for the development of assisted reproduction techniques for rare and endangered amphibians, to short-term storage at temperatures > 0 degrees C were studied. Whole excised testes were stored at 0 degrees or 4 degrees C for 15 days, and sperm motility was measured at excision and after storage for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed > 50% motility for 7 days at 0 degrees C and for 5 days at 4 degrees C. At 15 days, only spermatozoa stored at 0 degrees C still showed some motility (3%). Sperm suspensions were prepared at 5 day intervals over 30 days in simplified amphibian ringer (SAR) at dilutions of 1:1, 1:5 and 1:10 (w/v) testes:SAR. Aliquots from each dilution were stored at 0 degrees C in Eppendorf tubes opened at 5 day intervals of storage (aerated) or kept sealed (unaerated) (treatments: aerated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). After 30 days, sperm motility and fertilizing capacity were determined. The optimal protocol for sperm storage up to 10 days, as assessed by the retention of fertilizing capacity, was as a 1:5 testis:SAR (w/v) suspension, whereas the longest absolute retention of both motility and fertilizing capacity was observed in concentrated (1:1 dilution), anaerobic suspensions (up to 25-30 days). Oviductal oocytes placed in SAR at 5, 10, 15, 20 and 25 degrees C immediately after ovulation lost viability when cooled rapidly to 5 degrees C and stored for 2 h. However, oocytes retained viability for up to 8 h at the optimum storage temperature of 15 degrees C. Thus, it is concluded that during short-term storage spermatozoa retain viability for longer than oocytes, and that spermatozoa in suspensions retain viability for longer than spermatozoa stored in situ in excised testes.     

Effect of local heating of rat testes after suppression of spermatogenesis by pretreatment with a GnRH agonist and an anti-androgen

The effects of local heating of rat testes, in which spermatogenesis had been suppressed with injections of a GnRH agonist and an anti-androgen, were examined. Although the detrimental effects of heating were not as marked as those found in the testes of non-injected rats, the testes in which spermatogenesis was suppressed also showed a significant reduction in mass, the number of spermatozoa, tubular diameter and the percentage of normal tubular cross-sections at day 35 after heating. The results indicate that heating has an effect on cells in the testis other than those shown to be most susceptible to heat, namely pachytene spermatocytes and early spermatids, which were absent or markedly reduced in number when spermatogenesis was suppressed. The long-term effects of heating on the above parameters, as reported in a previous study, were also confirmed. However, in testes in which spermatogenesis was suppressed at the time of heating, there appeared to be no or a reduced long-term impairment of spermatogenesis, as determined by testis mass, the percentage of qualitatively normal tubules and epididymal sperm counts.     

Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations,activation and development of mouse oocytes

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.     

Paternal effect on embryo quality in diabetic mice is related to poor sperm quality and associated with decreased glucose transporter expression

The objective of this study was to determine whether sperm quality, fertilization capacity, and subsequent embryo development are altered in diabetic male mice and whether differences in facilitative glucose transporter (GLUT; now known as solute carrier family 2, SLC2A) expression in the testis and sperm exist. Using two type 1 diabetic mouse models, SLC2A expression in the testis and sperm was determined by western immunoblotting and immunofluorescence staining. To address sperm quality and fertilization capacity, computer-assisted sperm analysis and in vitro fertilization were performed. SLC2A1, SLC2A3, and SLC2A5 did not change in expression in the testes or sperm between diabetic and non-diabetic mice. SLC2A8 and SLC2A9b were less expressed in the testes of both diabetic models versus controls. SLC2A9a was not expressed in the Akita testis or sperm when compared with strain-matched controls. 3beta-hydroxysteroid dehydrogenase (HSD3B) expression was significantly decreased in the Leydig cells from the diabetic mice. Sperm concentration and motility were significantly lower in both the diabetics when compared with the control. These parameters normalized in Akita diabetic males treated with insulin. In addition, fertilization rates were significantly lower in the Akita group (17.9%) and the streptozotocin (STZ)-injected male group (43.6%) when compared with the normal group (88.8%). Interestingly, of the fertilized zygotes, embryo developmental rates to the blastocyst stage were lower in both diabetic models (7.1% Akita and 50.0% STZ) when compared with controls (71.7%). Male diabetes may cause male subfertility by altering steroidogenesis, sperm motility, and SLC2A expression. This is the first study to link a paternal metabolic abnormality to a sperm effect on cell division and subsequent embryonic development.     

A quantitative (stereological) study of the effects of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis

The aim of this study was to examine the controversial effects of experimental unilateral cryptorchidism and subsequent orchiopexy on the number of germ cells and other morphometric characteristics of testicular and epididymal structures in adult rabbits. Unilateral cryptorchidism was induced in 11 mature male New Zealand white rabbits by returning one testis, together with the ipsilateral epididymis, to the abdominal cavity via a surgical procedure. After 3 months, testes and epididymides were removed from six animals (and from six age-matched control animals that did not undergo the surgery). Orchiopexy was performed on the five remaining animals and the testes and epididymides of these animals (and an additional six age-matched control animals) were removed 7 weeks later. A contemporary, unbiased and efficient stereological tool, the optical disector, was used to estimate the number of nuclei in the testis and epididymis using methacrylate-embedded sections of 25 micron in thickness. Cryptorchidism resulted in severe testicular atrophy and spermatogenic arrest: type A spermatogonia and Sertoli cells only were seen in the seminiferous epithelium, and the number of type A spermatogonia per testis was reduced by 84%. After orchiopexy, the testis remained atrophied and the number of type A spermatogonia returned to the near-normal range in four of five animals, but spermatogenesis was recovered only partially at the stage of early primary spermatocytes (one animal), late primary spermatocytes (two animals) or spermatids (one animal). In conclusion, cryptorchidism caused severe spermatogenic arrest that was potentially recoverable (in view of the restoration of the number of type A spermatogonia), but orchiopexy failed to induce full recovery of spermatogenesis.     

A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size

The plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, show marked differences in the size of their testes and in the number of spermatozoa within the epididymides. In the present study, the dynamics of sperm production and the duration of sperm transit along the male excurrent ducts were compared between these two species. The durations of the cycle of the seminiferous epithelium, spermatogenesis and sperm transit were determined by tracking cells using autoradiography after [(3)H]thymidine incorporation. Daily sperm production was determined from counts of testicular spermatids after homogenization and further estimates of sperm transit were obtained by dividing sperm reserves within the various regions of the extratesticular ducts by the daily sperm production of the attached testis. In the plains rat, the mean duration of the cycle of the seminiferous epithelium was 11.2 days, the duration of spermatogenesis was 45 days, daily sperm production was 2.6 x 10(7) spermatozoa per gram of testis and epididymal transit of spermatozoa took approximately 9 days (caput 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in the hopping mouse, the mean duration of the cycle of the seminiferous epithelium was 14 days, the duration of spermatogenesis was 56 days and daily sperm production per gram of testis was < 1.0 x 10(7). Epididymal transit of spermatozoa was completed in about 4 days (caput + corpus < 1 day; cauda 3 days); however, spermatozoa may be stored for an additional 1.5-2.0 days in the vas deferens. These results indicate that, in addition to small testes, the hopping mouse shows a low efficiency of sperm production, a relatively long duration of spermatogenesis and rapid passage of spermatozoa through the epididymis, all of which contribute to low epididymal sperm counts. These data are considered in relation to interspecific differences in sperm competition.     

Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase Czeta

The molecular mechanism by which sperm triggers Ca2+ oscillation, oocyte activation, and early embryonic development has not been clarified. Recently, oocyte activation has been shown to be induced by sperm-specific phospholipase Czeta (PLCzeta). The ability of PLCzeta to induce oocyte activation is highly conserved across vertebrates. In the present study, porcine PLCzeta cDNA was identified and the nucleotide sequence was determined. The expression pattern of porcine PLCzeta mRNA during the period of postnatal testicular development was shown to be similar to that of mouse PLCzeta. PLCzeta mRNA expression in the pig and mouse was detected only in the testes when the elongated spermatids had differentiated, and was detected from day 96 after birth in the pig. Histological examination of porcine testis during the period of postnatal development revealed the presence of spermatozoa from day 110 after birth. These findings suggest that the synthesis of PLCzeta mRNA starts when spermiogenesis is initiated. Microinjection of porcine PLCzeta complementary RNA into porcine oocytes demonstrated that porcine PLCzeta has the ability to trigger repetitive Ca2+ transients in porcine oocytes similar to that observed during fertilization. It was also found that porcine PLCzeta cRNA has the potential to induce oocyte activation and initiate embryonic development up to the blastocyst stage.     

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.